lipofectamine 2000  (Thermo Fisher)


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    Name:
    Lipofectamine 2000 Transfection Reagent
    Description:
    Lipofectamine 2000 Transfection Reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines Researchers use Lipofectamine 2000 Reagent for siRNA and shRNA based gene knockdown experiments as well as for gene expression studies With Lipofectamine 2000 Transfection Reagent you ll get • Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression see table • Superior performance for co transfection of siRNA and plasmid DNA• Proven efficacy in the presence of serum eliminates the need to change media following transfection• Reliable performance for high throughput applications• The best choice for establishing stable cell linesA high performance transfection reagent for gene expression and gene silencingLipofectamine 2000 Transfection Reagent works effectively with all common cell lines as well as with many challenging ones and can be used in media with or without serum For gene silencing Lipofectamine 2000 Transfection Reagent s high efficiency transfections provide the high levels of gene knockdown needed to produce convincing results Lipofectamine 2000 Transfection Reagent is the number one choice for co transfection given its effectiveness for transfecting both siRNA and plasmid DNA Lipofectamine 2000 Transfection Reagent is easy to use simply mix with nucleic acid and add to cell culture Ideal for high throughput workSimplicity and speed combined with high transfection efficiency make Lipofectamine 2000 Transfection Reagent ideal for transient protein expression or high throughput RNAi transfections Transfection conditions can be easily established for automated or robotic systems used in such applications
    Catalog Number:
    11668019
    Price:
    None
    Category:
    Cell Culture Transfection Reagents
    Applications:
    Cell Culture|Plasmid Transfection|RNAi Transfection|Stem Cell & Primary Cell Transfections|Synthetic siRNA Transfection|shRNA & miR RNAi Plasmid Transfection|Transfection
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    Structured Review

    Thermo Fisher lipofectamine 2000
    XPD monitored the related genes expression. A431 cells were transfected with the same amount of medium (Control), <t>Lipofectamine</t> (LF), pEGFP-N2 (EM) + Lipofectamine (EM+LF), or pEGFP-N2-XPD + Lipofectamine (XPD+LF). ( A ) The overexpression of XPD affected the mRNA levels of related genes for cell proliferation and cell apoptosis. After cell transfection, cells were collected and total RNA was isolated for qRT-PCR. ( B ) The effect of XPD overexpression on related proteins expression was analyzed by Western blot. ( C ) The target bands were normalized to β-actin using Quantity One software. * P
    Lipofectamine 2000 Transfection Reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines Researchers use Lipofectamine 2000 Reagent for siRNA and shRNA based gene knockdown experiments as well as for gene expression studies With Lipofectamine 2000 Transfection Reagent you ll get • Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression see table • Superior performance for co transfection of siRNA and plasmid DNA• Proven efficacy in the presence of serum eliminates the need to change media following transfection• Reliable performance for high throughput applications• The best choice for establishing stable cell linesA high performance transfection reagent for gene expression and gene silencingLipofectamine 2000 Transfection Reagent works effectively with all common cell lines as well as with many challenging ones and can be used in media with or without serum For gene silencing Lipofectamine 2000 Transfection Reagent s high efficiency transfections provide the high levels of gene knockdown needed to produce convincing results Lipofectamine 2000 Transfection Reagent is the number one choice for co transfection given its effectiveness for transfecting both siRNA and plasmid DNA Lipofectamine 2000 Transfection Reagent is easy to use simply mix with nucleic acid and add to cell culture Ideal for high throughput workSimplicity and speed combined with high transfection efficiency make Lipofectamine 2000 Transfection Reagent ideal for transient protein expression or high throughput RNAi transfections Transfection conditions can be easily established for automated or robotic systems used in such applications
    https://www.bioz.com/result/lipofectamine 2000/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    lipofectamine 2000 - by Bioz Stars, 2021-05
    99/100 stars

    Images

    1) Product Images from "Role of Xeroderma Pigmentosum Group D in Cell Cycle and Apoptosis in Cutaneous Squamous Cell Carcinoma A431 Cells"

    Article Title: Role of Xeroderma Pigmentosum Group D in Cell Cycle and Apoptosis in Cutaneous Squamous Cell Carcinoma A431 Cells

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.905319

    XPD monitored the related genes expression. A431 cells were transfected with the same amount of medium (Control), Lipofectamine (LF), pEGFP-N2 (EM) + Lipofectamine (EM+LF), or pEGFP-N2-XPD + Lipofectamine (XPD+LF). ( A ) The overexpression of XPD affected the mRNA levels of related genes for cell proliferation and cell apoptosis. After cell transfection, cells were collected and total RNA was isolated for qRT-PCR. ( B ) The effect of XPD overexpression on related proteins expression was analyzed by Western blot. ( C ) The target bands were normalized to β-actin using Quantity One software. * P
    Figure Legend Snippet: XPD monitored the related genes expression. A431 cells were transfected with the same amount of medium (Control), Lipofectamine (LF), pEGFP-N2 (EM) + Lipofectamine (EM+LF), or pEGFP-N2-XPD + Lipofectamine (XPD+LF). ( A ) The overexpression of XPD affected the mRNA levels of related genes for cell proliferation and cell apoptosis. After cell transfection, cells were collected and total RNA was isolated for qRT-PCR. ( B ) The effect of XPD overexpression on related proteins expression was analyzed by Western blot. ( C ) The target bands were normalized to β-actin using Quantity One software. * P

    Techniques Used: Expressing, Transfection, Over Expression, Isolation, Quantitative RT-PCR, Western Blot, Software

    XPD repressed cell proliferation. A431 cells were divided into 4 groups, and the control group was administered the same amount of medium (Control group). The other 3 groups were transfected with Lipofectamine (LF group), pEGFP-N2 (empty vector) + Lipofectamine (EM+LF group), or pEGFP-N2-XPD + Lipofectamine (XPD+LF group). ( A ) The signals of XPD were detected after pEGFP-N2-XPD transfection. After cell transfection, A431 cells were observed with an inverted fluorescence microscope. Scale bar=100 μm. ( B ) The mRNA level of XPD was increased in the XPD+LF group. Total RNA was isolated from 4 groups for QRT-PCR analysis. * P
    Figure Legend Snippet: XPD repressed cell proliferation. A431 cells were divided into 4 groups, and the control group was administered the same amount of medium (Control group). The other 3 groups were transfected with Lipofectamine (LF group), pEGFP-N2 (empty vector) + Lipofectamine (EM+LF group), or pEGFP-N2-XPD + Lipofectamine (XPD+LF group). ( A ) The signals of XPD were detected after pEGFP-N2-XPD transfection. After cell transfection, A431 cells were observed with an inverted fluorescence microscope. Scale bar=100 μm. ( B ) The mRNA level of XPD was increased in the XPD+LF group. Total RNA was isolated from 4 groups for QRT-PCR analysis. * P

    Techniques Used: Transfection, Plasmid Preparation, Fluorescence, Microscopy, Isolation, Quantitative RT-PCR

    The overexpression of XPD promoted cell apoptosis. A431 cells were transfected with the same amount of medium (Control, A ), Lipofectamine (LF, B ), pEGFP-N2 (EM) + Lipofectamine (EM+LF, C ), or pEGFP-N2-XPD + Lipofectamine (XPD+LF, D ). Cells that stained positive for early apoptosis markers (annexin V-FITC stained only) and for late apoptosis markers (annexin V-FITC and PI stained) were combined for analysis.
    Figure Legend Snippet: The overexpression of XPD promoted cell apoptosis. A431 cells were transfected with the same amount of medium (Control, A ), Lipofectamine (LF, B ), pEGFP-N2 (EM) + Lipofectamine (EM+LF, C ), or pEGFP-N2-XPD + Lipofectamine (XPD+LF, D ). Cells that stained positive for early apoptosis markers (annexin V-FITC stained only) and for late apoptosis markers (annexin V-FITC and PI stained) were combined for analysis.

    Techniques Used: Over Expression, Transfection, Staining

    2) Product Images from "BMCC1 Is an AP-2 Associated Endosomal Protein in Prostate Cancer Cells"

    Article Title: BMCC1 Is an AP-2 Associated Endosomal Protein in Prostate Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073880

    Cellular distribution of BMCC1. A. BMCC1 distribution in LNCaP . BMCC1 was labelled by immunostaining with rabbit anti-BMCC1 antibodies to the C- or N- (Ab1, Ab2) terminus or non-specific (NS) rabbit antibody and detected with donkey anti-rabbit Alexafluor 594. Cells were counterstained with Hoechst. Staining was analysed by wide-field fluorescent microscopy (x63). B. High resolution imaging of BMCC1 in LNCaP . Fixed LNCaP were incubated with a rabbit anti-BMCC1 (Ab-1) and a sheep anti-BMCC1 (Ab-4). Cells were stained with donkey anti-rabbit Alexafluor 488 and donkey anti-sheep Alexafluor 594. Staining was analysed by confocal fluorescent microscopy. C. Ablation of BMCC1 immunostaining by siRNA . LNCaP were transfected with BMCC1 siRNA 1730 (siBMCC1) or control siRNA (siControl) using Lipofectamine RNAiMAX. Cells were fixed 3 days post transfection and stained with rabbit anti-BMCC1 (Ab-1) and detected using donkey anti-rabbit Alexafluor 594. D. Subcellular fractionation of LNCaP . LNCaP cytoplasmic extract was generated by dounce homogenization in hypotonic solution and sedimentation of nuclei. Microsomes were precipitated from the recovered cytoplasm by ultracentrifugation. BMCC1 compartmentalization was analysed by western blotting the resulting microsomal and cytosolic fractions using antibodies against BMCC1 (Ab-4) (5% gel), β-adaptin and α-tubulin (microsomes) and GAPDH (cytosolic) (duplicate 12% gel).
    Figure Legend Snippet: Cellular distribution of BMCC1. A. BMCC1 distribution in LNCaP . BMCC1 was labelled by immunostaining with rabbit anti-BMCC1 antibodies to the C- or N- (Ab1, Ab2) terminus or non-specific (NS) rabbit antibody and detected with donkey anti-rabbit Alexafluor 594. Cells were counterstained with Hoechst. Staining was analysed by wide-field fluorescent microscopy (x63). B. High resolution imaging of BMCC1 in LNCaP . Fixed LNCaP were incubated with a rabbit anti-BMCC1 (Ab-1) and a sheep anti-BMCC1 (Ab-4). Cells were stained with donkey anti-rabbit Alexafluor 488 and donkey anti-sheep Alexafluor 594. Staining was analysed by confocal fluorescent microscopy. C. Ablation of BMCC1 immunostaining by siRNA . LNCaP were transfected with BMCC1 siRNA 1730 (siBMCC1) or control siRNA (siControl) using Lipofectamine RNAiMAX. Cells were fixed 3 days post transfection and stained with rabbit anti-BMCC1 (Ab-1) and detected using donkey anti-rabbit Alexafluor 594. D. Subcellular fractionation of LNCaP . LNCaP cytoplasmic extract was generated by dounce homogenization in hypotonic solution and sedimentation of nuclei. Microsomes were precipitated from the recovered cytoplasm by ultracentrifugation. BMCC1 compartmentalization was analysed by western blotting the resulting microsomal and cytosolic fractions using antibodies against BMCC1 (Ab-4) (5% gel), β-adaptin and α-tubulin (microsomes) and GAPDH (cytosolic) (duplicate 12% gel).

    Techniques Used: Immunostaining, Staining, Microscopy, Imaging, Incubation, Transfection, Fractionation, Generated, Homogenization, Sedimentation, Western Blot

    3) Product Images from "Lentinan from shiitake selectively attenuates AIM2 and non-canonical inflammasome activation while inducing pro-inflammatory cytokine production"

    Article Title: Lentinan from shiitake selectively attenuates AIM2 and non-canonical inflammasome activation while inducing pro-inflammatory cytokine production

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01462-4

    Effect of lentinan on endotoxemic models. ( A ) Mice (n = 10 per group) were intraperitoneally (ip) injected with LPS (25 mg/kg) and/or lentinan (LNT) as indicated. Survival rates were observed at the indicated times. ( B ), LPS-primed BMDMs were transfected with LPS to activate non-canonical inflammasome in the presence of LNT as indicated. IL-1β and Casp1 secretion was analyzed by ELISA and immunoblotting. ( B ) LPS-primed BMDMs were infected with E . coli . (MOI = 10) to activate non-canonical inflammasome in the presence of LNT as indicated. Secretion of Casp1 was analyzed by immunoblotting, and the right bar graph indicates relative band density. Bar graphs present the mean ± SD. Lipo, Lipofectamine 2000 TM .
    Figure Legend Snippet: Effect of lentinan on endotoxemic models. ( A ) Mice (n = 10 per group) were intraperitoneally (ip) injected with LPS (25 mg/kg) and/or lentinan (LNT) as indicated. Survival rates were observed at the indicated times. ( B ), LPS-primed BMDMs were transfected with LPS to activate non-canonical inflammasome in the presence of LNT as indicated. IL-1β and Casp1 secretion was analyzed by ELISA and immunoblotting. ( B ) LPS-primed BMDMs were infected with E . coli . (MOI = 10) to activate non-canonical inflammasome in the presence of LNT as indicated. Secretion of Casp1 was analyzed by immunoblotting, and the right bar graph indicates relative band density. Bar graphs present the mean ± SD. Lipo, Lipofectamine 2000 TM .

    Techniques Used: Mouse Assay, Injection, Transfection, Enzyme-linked Immunosorbent Assay, Infection

    Related Articles

    Molecular Weight:

    Article Title: Enhanced mRNA delivery into lymphocytes enabled by lipid-varied libraries of charge-altering releasable transporters
    Article Snippet: .. Regenerated cellulose dialysis membranes (Spectra/Por 6 Standard RC; molecular weight cutoff 1,000) were purchased from Spectrum Laboratories, Inc. Lipofectamine 2000 was purchased from Life Technologies. .. MTT was purchased from Fluka.

    Incubation:

    Article Title: Innovative approaches to genome editing in avian species
    Article Snippet: Preliminary results indicate that using the STAGE protocol for sperm preparation with quail, chickens, and turkeys leads to successful transfection of RNA. .. Sperm was washed and then incubated with Lipofectamine® 2000 and a fluorescently labeled RNA (BLOCK-iT™, Thermo Fisher). .. The results indicate that sperm from all three species remained motile during the transfection process and that the RNA was effectively delivered to the sperm (Fig. ).

    Labeling:

    Article Title: Innovative approaches to genome editing in avian species
    Article Snippet: Preliminary results indicate that using the STAGE protocol for sperm preparation with quail, chickens, and turkeys leads to successful transfection of RNA. .. Sperm was washed and then incubated with Lipofectamine® 2000 and a fluorescently labeled RNA (BLOCK-iT™, Thermo Fisher). .. The results indicate that sperm from all three species remained motile during the transfection process and that the RNA was effectively delivered to the sperm (Fig. ).

    Blocking Assay:

    Article Title: Innovative approaches to genome editing in avian species
    Article Snippet: Preliminary results indicate that using the STAGE protocol for sperm preparation with quail, chickens, and turkeys leads to successful transfection of RNA. .. Sperm was washed and then incubated with Lipofectamine® 2000 and a fluorescently labeled RNA (BLOCK-iT™, Thermo Fisher). .. The results indicate that sperm from all three species remained motile during the transfection process and that the RNA was effectively delivered to the sperm (Fig. ).

    Article Title: Endothelial cell-specific chemotaxis receptor (ecscr) promotes angioblast migration during vasculogenesis and enhances VEGF receptor sensitivity
    Article Snippet: Transfection of both cell types was performed by Lipofection (Invitrogen). .. ECSCR siRNA was generated using the Dicer method using the Block-iT kit from Invitrogen. siRNA transfection of human umbilical vein endothelial cells (HUVECs) was performed at 80% confluence using 500 ng of siRNA and Lipofectamine 2000 Reagent (Invitrogen). ..

    Transfection:

    Article Title: VBP1 facilitates proteasome and autophagy-mediated degradation of MutS homologue hMSH4
    Article Snippet: Most plasmid DNA transfections were carried out using standard calcium phosphate method. .. The siRNA transfections were carried out by the use of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. .. VBP1 sh-2 was transfected into HEK293T cells using Amaxa Nucleofector (Lonza Group, Basel, Switzerland).

    Article Title: KDM5 histone demethylases repress immune response via suppression of STING
    Article Snippet: SKBR3 cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. siRNA transfections were performed using RNAiMAX (Invitrogen), and plasmid transfections were performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. .. The sequence of dsDNA90 was described previously [ ], transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. siRNA universal negative control 1 and 2 were purchased from Sigma (SIC001 and SIC002). siKDM5A targeting sequences were described previously [ ]. ..

    Article Title: Functional MicroRNA Library Screening Identifies the HypoxaMiR MiR-24 as a Potent Regulator of Smooth Muscle Cell Proliferation and Vascularization
    Article Snippet: The cell culture medium was changed after 48 h and consisted of DMEM (Biochrom F0415), 10% horse serum (Gibco 26050), 2% chick embryo extract, 1% penicillin/streptomycin (Gibco 15140), insulin (10 μg/ml; Sigma I9278), and aprotinin (33 μg/ml; Sigma A1153). .. Transfection of VE-CadherinxRosaR-26 EHTs was performed with Lipofectamine™ 2000 Reagent (Invitrogen) according to the manufacturer's instructions. .. The amount of miRNA and transfection agent was adapted to the use for the EHT culture format.

    Article Title: Reprogramming fibroblasts to pluripotency using arginine-terminated polyamidoamine nanoparticles based non-viral gene delivery system
    Article Snippet: The following day, the culture medium was replaced with solutions of G4Arg-DNA complexes at various N/P ratios with 5 μg of plasmid. .. Lipofectamine® 2000 (Invitrogen® , Life Technologies Corp) -based gene transfection was performed as a positive control, according to the manufacturer’s protocol. .. After incubation at 37°C for 4 hours, the transfection medium was replaced with fresh fibroblast medium.

    Article Title: Endothelial cell-specific chemotaxis receptor (ecscr) promotes angioblast migration during vasculogenesis and enhances VEGF receptor sensitivity
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    Sequencing:

    Article Title: KDM5 histone demethylases repress immune response via suppression of STING
    Article Snippet: SKBR3 cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. siRNA transfections were performed using RNAiMAX (Invitrogen), and plasmid transfections were performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. .. The sequence of dsDNA90 was described previously [ ], transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. siRNA universal negative control 1 and 2 were purchased from Sigma (SIC001 and SIC002). siKDM5A targeting sequences were described previously [ ]. ..

    Negative Control:

    Article Title: KDM5 histone demethylases repress immune response via suppression of STING
    Article Snippet: SKBR3 cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. siRNA transfections were performed using RNAiMAX (Invitrogen), and plasmid transfections were performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. .. The sequence of dsDNA90 was described previously [ ], transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. siRNA universal negative control 1 and 2 were purchased from Sigma (SIC001 and SIC002). siKDM5A targeting sequences were described previously [ ]. ..

    Positive Control:

    Article Title: Reprogramming fibroblasts to pluripotency using arginine-terminated polyamidoamine nanoparticles based non-viral gene delivery system
    Article Snippet: The following day, the culture medium was replaced with solutions of G4Arg-DNA complexes at various N/P ratios with 5 μg of plasmid. .. Lipofectamine® 2000 (Invitrogen® , Life Technologies Corp) -based gene transfection was performed as a positive control, according to the manufacturer’s protocol. .. After incubation at 37°C for 4 hours, the transfection medium was replaced with fresh fibroblast medium.

    Generated:

    Article Title: Endothelial cell-specific chemotaxis receptor (ecscr) promotes angioblast migration during vasculogenesis and enhances VEGF receptor sensitivity
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  • 97
    Thermo Fisher cell transfections
    Nuclear FGFR1 mediates NGF induced neurite outgrowth and activation of the th gene promoter. ( A ) PC12 cells were transfected with two plasmids, one expressing recombinant FGFR1 or control pcDNA3.1 and the second expressing EGFP. EGFP diffuses throughout the cell permitting visualization of the entire neuritic network. More than 90% of cells co-expressed transfected plasmids as reported in previous studies [29] , [73] . Twenty-four hours after <t>transfection</t> cultures were switched to 1% horse serum medium with or without (control) 50 ng/ml NGF for an additional 10 days, after which the cells were imaged using fluorescent microscopy. Bar length - 100 µm. ( B ) The longest process in an individual transfected cell was measured using ImageJ [24] .* mark comparison to pCDNA3.1 (-NGF) and # to pCNA3.1 (+NGF). Transfection of constitutive nuclear FGFR1(SP−/NLS) increased neurite outgrowth approximately 3-fold (*p
    Cell Transfections, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cell transfections/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cell transfections - by Bioz Stars, 2021-05
    97/100 stars
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    Image Search Results


    Nuclear FGFR1 mediates NGF induced neurite outgrowth and activation of the th gene promoter. ( A ) PC12 cells were transfected with two plasmids, one expressing recombinant FGFR1 or control pcDNA3.1 and the second expressing EGFP. EGFP diffuses throughout the cell permitting visualization of the entire neuritic network. More than 90% of cells co-expressed transfected plasmids as reported in previous studies [29] , [73] . Twenty-four hours after transfection cultures were switched to 1% horse serum medium with or without (control) 50 ng/ml NGF for an additional 10 days, after which the cells were imaged using fluorescent microscopy. Bar length - 100 µm. ( B ) The longest process in an individual transfected cell was measured using ImageJ [24] .* mark comparison to pCDNA3.1 (-NGF) and # to pCNA3.1 (+NGF). Transfection of constitutive nuclear FGFR1(SP−/NLS) increased neurite outgrowth approximately 3-fold (*p

    Journal: PLoS ONE

    Article Title: NGF-Induced Cell Differentiation and Gene Activation Is Mediated by Integrative Nuclear FGFR1 Signaling (INFS)

    doi: 10.1371/journal.pone.0068931

    Figure Lengend Snippet: Nuclear FGFR1 mediates NGF induced neurite outgrowth and activation of the th gene promoter. ( A ) PC12 cells were transfected with two plasmids, one expressing recombinant FGFR1 or control pcDNA3.1 and the second expressing EGFP. EGFP diffuses throughout the cell permitting visualization of the entire neuritic network. More than 90% of cells co-expressed transfected plasmids as reported in previous studies [29] , [73] . Twenty-four hours after transfection cultures were switched to 1% horse serum medium with or without (control) 50 ng/ml NGF for an additional 10 days, after which the cells were imaged using fluorescent microscopy. Bar length - 100 µm. ( B ) The longest process in an individual transfected cell was measured using ImageJ [24] .* mark comparison to pCDNA3.1 (-NGF) and # to pCNA3.1 (+NGF). Transfection of constitutive nuclear FGFR1(SP−/NLS) increased neurite outgrowth approximately 3-fold (*p

    Article Snippet: Cell transfections were performed using Lipofectamine 2000 (Invitrogen).

    Techniques: Activation Assay, Transfection, Expressing, Recombinant, Microscopy

    NGF stimulates FGFR1 binding to NGF-activated genes and cooperates with FGFR1 in activation of Nur-responsive elements - NuRE and NBRE. ( A ) In vivo validation of FGFR1 and Nur binding to an NBRE-like site within intron 1 of the DCX gene. Chromatin was immunoprecipitated with FGFR1, Nur77/Nurr1Ab or IgG in dissected rat brain regions including, the olfactory bulb (OB) which express the dcx gene at high levels [62] , and in the cortex (CX), cerebellum (CB) and ventral midbrain (VM), which express the dcx gene at lower levels. The cyclophilin A gene, which lacks potential Nur-binding NurRE, NBRE or RARE like elements was used as a control. Samples were combined from two rats and the graphs show ΔΔCt means±SEM of triplicate assays. ( B ) PC 12 cells were incubated with or without NGF for 48 hours. ChIP-qPCR was performed with FGFR1 or Nur77/Nurr1 antibodies or control IgG within diverse NGF-activated genes. Similar as in vivo, no binding to the cyclophilinA gene was observed (not shown). Graphs show means of duplicate or triplicate samples from a representative experiment. Similar changes were observed in three separate experiments. ( C,D ) FGFR1 augments Nur77 NurRE- and NBRE-dependent transcription in PC12 cells. Cells were transfected with NurRE-Luc ( C ) or NBRE-Luc ( D ) and either FGFR1(SP−/NLS) or dominant negative FGFR1(TK-) in the presence or absence of Nur77. The amount of transfected DNA was adjusted to 1 µg per well. One day after transfection, PC12 cells were incubated with NGF (50 ng/ml) or without NGF for an additional 24 hours. Results represent the mean ± SEM from 2 experiments with at least three replicates. One-Way ANOVA, LSD: *p

    Journal: PLoS ONE

    Article Title: NGF-Induced Cell Differentiation and Gene Activation Is Mediated by Integrative Nuclear FGFR1 Signaling (INFS)

    doi: 10.1371/journal.pone.0068931

    Figure Lengend Snippet: NGF stimulates FGFR1 binding to NGF-activated genes and cooperates with FGFR1 in activation of Nur-responsive elements - NuRE and NBRE. ( A ) In vivo validation of FGFR1 and Nur binding to an NBRE-like site within intron 1 of the DCX gene. Chromatin was immunoprecipitated with FGFR1, Nur77/Nurr1Ab or IgG in dissected rat brain regions including, the olfactory bulb (OB) which express the dcx gene at high levels [62] , and in the cortex (CX), cerebellum (CB) and ventral midbrain (VM), which express the dcx gene at lower levels. The cyclophilin A gene, which lacks potential Nur-binding NurRE, NBRE or RARE like elements was used as a control. Samples were combined from two rats and the graphs show ΔΔCt means±SEM of triplicate assays. ( B ) PC 12 cells were incubated with or without NGF for 48 hours. ChIP-qPCR was performed with FGFR1 or Nur77/Nurr1 antibodies or control IgG within diverse NGF-activated genes. Similar as in vivo, no binding to the cyclophilinA gene was observed (not shown). Graphs show means of duplicate or triplicate samples from a representative experiment. Similar changes were observed in three separate experiments. ( C,D ) FGFR1 augments Nur77 NurRE- and NBRE-dependent transcription in PC12 cells. Cells were transfected with NurRE-Luc ( C ) or NBRE-Luc ( D ) and either FGFR1(SP−/NLS) or dominant negative FGFR1(TK-) in the presence or absence of Nur77. The amount of transfected DNA was adjusted to 1 µg per well. One day after transfection, PC12 cells were incubated with NGF (50 ng/ml) or without NGF for an additional 24 hours. Results represent the mean ± SEM from 2 experiments with at least three replicates. One-Way ANOVA, LSD: *p

    Article Snippet: Cell transfections were performed using Lipofectamine 2000 (Invitrogen).

    Techniques: Binding Assay, Activation Assay, In Vivo, Immunoprecipitation, Incubation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Dominant Negative Mutation

    NGF accelerates nuclear trafficking of FGFR1 by reducing FGFR1 nuclear export. ( A ) FGFR1-EGFP was transfected into PC12 cells. Twenty-four hours after transfection, cultures were treated with 50 ng/ml NGF and LMB (100 ng/ml in 0.1% v/v ethanol) or ethanol alone (0.1% v/v ethanol) for an additional 48 h and during subsequent imaging. Examples of FGFR1-EGFP expressing cells before and after photo-bleaching are shown. DIC image indicates the nuclear and cytoplasmic regions. About 1/3 nuclear area of PC12 cell was bleached by high intensity laser and 2–3 regions of interest (ROI) intensity were measured. ( B ) Single-exponential analysis of FGFR1-EGFP FLIP regression in cytoplasm and nucleus showed that the diffusion rate of FGFR1-EGFP is affected by NGF treatment in live cells. Individual curves represent means of at least 23 cells. NGF significantly increases the FGFR1-EGFP exchange between nucleus and cytoplasm (half-time decreases) without affecting the FGFR1-EGFP mobile population. ( C ) Single-exponential analysis of FGFR1-EGFP FLIP regression in the cytoplasm shows that NGF facilitates FGFR1-EGFP trafficking between the cytoplasm and nucleus (half-time decreases from 121.5 sec to 89.7 sec; One-way AVOVA, LSD*p

    Journal: PLoS ONE

    Article Title: NGF-Induced Cell Differentiation and Gene Activation Is Mediated by Integrative Nuclear FGFR1 Signaling (INFS)

    doi: 10.1371/journal.pone.0068931

    Figure Lengend Snippet: NGF accelerates nuclear trafficking of FGFR1 by reducing FGFR1 nuclear export. ( A ) FGFR1-EGFP was transfected into PC12 cells. Twenty-four hours after transfection, cultures were treated with 50 ng/ml NGF and LMB (100 ng/ml in 0.1% v/v ethanol) or ethanol alone (0.1% v/v ethanol) for an additional 48 h and during subsequent imaging. Examples of FGFR1-EGFP expressing cells before and after photo-bleaching are shown. DIC image indicates the nuclear and cytoplasmic regions. About 1/3 nuclear area of PC12 cell was bleached by high intensity laser and 2–3 regions of interest (ROI) intensity were measured. ( B ) Single-exponential analysis of FGFR1-EGFP FLIP regression in cytoplasm and nucleus showed that the diffusion rate of FGFR1-EGFP is affected by NGF treatment in live cells. Individual curves represent means of at least 23 cells. NGF significantly increases the FGFR1-EGFP exchange between nucleus and cytoplasm (half-time decreases) without affecting the FGFR1-EGFP mobile population. ( C ) Single-exponential analysis of FGFR1-EGFP FLIP regression in the cytoplasm shows that NGF facilitates FGFR1-EGFP trafficking between the cytoplasm and nucleus (half-time decreases from 121.5 sec to 89.7 sec; One-way AVOVA, LSD*p

    Article Snippet: Cell transfections were performed using Lipofectamine 2000 (Invitrogen).

    Techniques: Transfection, Imaging, Expressing, Diffusion-based Assay, Size-exclusion Chromatography

    Hsp105 overexpression reduces CFTR synthesis but promotes its folding. A and B, HEK cells were co-transfected with wild-type ( A ) or ΔF508 ( B ) CFTR together with a control or Hsp105 ( 105 ) expression plasmid. Cells were lysed 24 h after transfection.

    Journal: The Journal of Biological Chemistry

    Article Title: Human Heat Shock Protein 105/110 kDa (Hsp105/110) Regulates Biogenesis and Quality Control of Misfolded Cystic Fibrosis Transmembrane Conductance Regulator at Multiple Levels *

    doi: 10.1074/jbc.M111.297580

    Figure Lengend Snippet: Hsp105 overexpression reduces CFTR synthesis but promotes its folding. A and B, HEK cells were co-transfected with wild-type ( A ) or ΔF508 ( B ) CFTR together with a control or Hsp105 ( 105 ) expression plasmid. Cells were lysed 24 h after transfection.

    Article Snippet: Cell transfection was performed using Lipofectamine 2000 (Invitrogen) or Effectine (Qiagen, Valencia, CA).

    Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation

    MAP3K3 promotes cell proliferation and the epithelial-mesenchymal transition process. (A) A reverse transcription-quantitative polymerase chain reaction assay demonstrated the efficiency of the overexpression plasmid of MAP3K3 in A549 and H460 cells. (B) The indicated transfection on A549 and H460 cellular viabilities was determined with a Cell Counting Kit-8 assay. A549 and H460 cells were transfected with the indicated combinations of pcDNA3 and MAP3K3 or pri-miR-505 and MAP3K3 or the control group. MAP3K3 overexpression promoted cell viability. NS vs. pri-miR-505 and MAP3K3; ** P

    Journal: International Journal of Molecular Medicine

    Article Title: miR-505 inhibits cell growth and EMT by targeting MAP3K3 through the AKT-NFκB pathway in NSCLC cells

    doi: 10.3892/ijmm.2018.4041

    Figure Lengend Snippet: MAP3K3 promotes cell proliferation and the epithelial-mesenchymal transition process. (A) A reverse transcription-quantitative polymerase chain reaction assay demonstrated the efficiency of the overexpression plasmid of MAP3K3 in A549 and H460 cells. (B) The indicated transfection on A549 and H460 cellular viabilities was determined with a Cell Counting Kit-8 assay. A549 and H460 cells were transfected with the indicated combinations of pcDNA3 and MAP3K3 or pri-miR-505 and MAP3K3 or the control group. MAP3K3 overexpression promoted cell viability. NS vs. pri-miR-505 and MAP3K3; ** P

    Article Snippet: Cell transfection was performed by Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols.

    Techniques: Real-time Polymerase Chain Reaction, Over Expression, Plasmid Preparation, Transfection, Cell Counting

    14-3-3 protein controls proper SAC inactivation in a γ-subtype-dependent manner. ( a ) Forty-eight hours after transfection with an indicated siRNA, HeLa cells were stained with anti-H3-pSer28 (red) and 4',6-diamidino-2-phenylindole (blue). Immunoblotting with anti-H3-pSer28 and anti-α-tubulin (loading control) are also shown in insets. ( b ) The effect of 14-3-3γ knockdown on cell-cycle progression after DTB synchronization. ( c ) Live-cell imaging analyses using H2B-GFP-expressing HeLa cells. Box-and-whisker plots show the elapsed time that cells spent in mitosis from nuclear envelope breakdown ( t =0) to anaphase onset ( n ≥60 from two independent experiments). ( d ) The effect of 14-3-3γ silencing on KT-associated BubR1. Scale bar, 10 μm. ( e, f ) The effects of BubR1 or Mad2 silencing on mitotic arrest induced by 14-3-3γ depletion. Mitotic indices are shown as mean±s.e.m. from three independent experiments ( n > 200 each). * P

    Journal: Nature Communications

    Article Title: PI 3-kinase-dependent phosphorylation of Plk1-Ser99 promotes association with 14-3-3? and is required for metaphase-anaphase transition

    doi: 10.1038/ncomms2879

    Figure Lengend Snippet: 14-3-3 protein controls proper SAC inactivation in a γ-subtype-dependent manner. ( a ) Forty-eight hours after transfection with an indicated siRNA, HeLa cells were stained with anti-H3-pSer28 (red) and 4',6-diamidino-2-phenylindole (blue). Immunoblotting with anti-H3-pSer28 and anti-α-tubulin (loading control) are also shown in insets. ( b ) The effect of 14-3-3γ knockdown on cell-cycle progression after DTB synchronization. ( c ) Live-cell imaging analyses using H2B-GFP-expressing HeLa cells. Box-and-whisker plots show the elapsed time that cells spent in mitosis from nuclear envelope breakdown ( t =0) to anaphase onset ( n ≥60 from two independent experiments). ( d ) The effect of 14-3-3γ silencing on KT-associated BubR1. Scale bar, 10 μm. ( e, f ) The effects of BubR1 or Mad2 silencing on mitotic arrest induced by 14-3-3γ depletion. Mitotic indices are shown as mean±s.e.m. from three independent experiments ( n > 200 each). * P

    Article Snippet: Plasmid transfection was performed with Lipofectamine LTX and PLUS Reagents according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA).

    Techniques: Transfection, Staining, Live Cell Imaging, Expressing, Whisker Assay